Risk factors for local recurrence after breast-conserving surgery.

BACKGROUND: It is not clear whether risk factors for local recurrence after breast-conserving surgery differ in women having surgery for in situ or invasive cancer. Furthermore, the Nottingham Prognostic Index (NPI) and Nottingham Histological Grade (NHG) have been little studied as determinants of local recurrence risk. METHOD: In a case-control study (491 cases and 1098 controls) nested within a cohort of 7502 women who had surgery for in situ or invasive cancer of the breast, patient characteristics, tumour characteristics and treatment-related variables were evaluated as risk factors for local recurrence. RESULTS: Multivariate conditional logistic regression analyses showed that age below 40 years, tumour multicentricity and an unclear or unknown surgical margin were significant risk factors for local recurrence. Radiotherapy to the breast and adjuvant hormone therapy were protective. Cancer in situ was not associated with a higher risk of local recurrence than invasive cancer (odds ratio 1.0, 95 per cent confidence interval 0.8 to 1.3). NHG and NPI were not helpful in determining risk of local recurrence. CONCLUSION: Margin status, age, tumour multicentricity, and use of radiotherapy and adjuvant hormone therapy were important determinants of risk of local recurrence. With the exception of surgical margin, variables related to the quality of surgical management did not predict risk of local recurrence.

Local recurrence in the breast after conservative surgery--a study of prognosis and prognostic factors in 391 women.

In a population-based cohort of 6613 women with invasive breast cancer, who had breast-conserving surgery between 1981 and 1990, 391 recurrences in the operated breast were identified. The main aim of this study was to examine the prognosis and prognostic factors in different subgroups of local recurrences, characterised by the time to recurrence, location of recurrence and previously given radiotherapy. The median follow-up for women who had a local recurrence was 7.9 years. The life-table estimates for breast cancer-specific survival in women with local recurrences were 84.5% (standard error (S.E.) 1.8) at 5 years and 70.9% (S.E. 2.7) at 10 years. The risk of breast cancer death was highest among women who had an early (

Consequences of axillary recurrence after conservative breast surgery.

BACKGROUND: The aim was to study the incidence, time course and prognosis of patients who developed axillary recurrence after breast-conserving surgery, and to evaluate possible risk factors for axillary recurrence and prognostic factors after axillary recurrence. METHODS: In a population-based cohort of 6613 women with invasive breast cancer who had breast-conserving surgery between 1981 and 1990, 92 recurrences in the ipsilateral axilla were identified. Risk factors for axillary recurrence were studied in a case-control study nested in the cohort, and late survival was documented in the women with axillary recurrence. RESULTS: The overall risk of axillary recurrence was 1.0 per cent at 5 years and 1.7 per cent at 10 years. The risk of axillary recurrence increased with tumour size (P = 0.033) and was highest in younger women (odds ratio (OR) 3.9 for women aged less than 40 years compared with those aged 50-59 years). Radiotherapy to the breast reduced the risk of axillary recurrence (OR 0.1 (95 per cent confidence interval 0.1 to 0.4)). The breast cancer-specific survival rate after axillary recurrence, as measured from primary treatment, was 78.0 per cent at 5 years and 52.3 per cent at 10 years. Tumour size and node status had a statistically significant effect on death from breast cancer. CONCLUSION: Axillary recurrence is rare, although more common in younger women with large tumours. Radiotherapy to the breast was protective. Tumour size and node status were the most important prognostic factors in women with axillary recurrence.

Time trends in the results of breast conservation in 4694 women.

In a population-based cohort of 4694 women with invasive breast cancer, operated upon with breast conserving surgery (BCS) in 1981--1990 and followed through to 1997, we studied how this technique had been adopted into clinical practice, especially with reference to the use of radiotherapy (RT). Our main aim was to see whether there was a drift in the risk of local recurrence and breast cancer death over time. During the 30,151 person-years of observation in the cohort, there were 582 local recurrences, 456 breast cancer deaths and 438 deaths due to other causes. Postoperative RT was given to 70.2%, but usage increased over the period. The women not receiving RT were mostly elderly, but also in women <70 years, 20.4% did not receive RT. The risk for local recurrence after RT were 7.6 and 17.8% at 5 and 10 years, respectively. Without RT, more than 30% had a local recurrence at 10 years. Thus, the choice not to irradiate failed to target women at a low risk. In a multivariate Cox analysis taking tumour size, nodal status, age at operation and RT into account, there was a trend for a higher risk of local recurrence in the later time period, relative hazard 1.5 (95% confidence interval (CI) 1.0--2.1). Corrected survival was 93.3 and 85.2% at 5 and 10 years, respectively.

Methylation of the p16(Ink4a) tumor suppressor gene 5'-CpG island in breast cancer.

Methylation of the p16(Ink4a) tumor suppressor gene 5'CpG island was analyzed in 104 primary breast cancer specimens using Southern blotting and methylation specific polymerase chain reaction (PCR) (MSP). Eight and four tumors, respectively, showed methylation, and all MSP positive tumors were detected by Southern blotting. To investigate possible methylation not detectable by these methods, bisulphite genomic sequencing was performed in 220 clones from 14 selected tumors. Absent methylation or methylation of single CpG dinucleotides prevailed in all tumors, but of the MSP positive tumors, three contained alleles with methylation of 31 or 32 of the 32 analyzed CpG dinucleotides in the island. Partially methylated alleles were also observed. In a group with low p16(Ink4a) expression determined by Western blotting, four randomly selected tumors contained several identical clones with methylation of 15 CpG dinucleotides by bisulphite genomic sequencing but with a methylation pattern that did not support detection by either Southern blotting or MSP, increasing the potential significance of p16(Ink4a) methylation in breast cancer.

Intratumoral variations in DNA ploidy and s-phase fraction in human breast cancer.

To study intratumoral DNA ploidy heterogeneity and S-phase fraction (SPF) variability, we prospectively collected five different samples from 48 breast carcinomas and each sample was analysed separately by flow cytometry. Aneuploidy rate was 89.6% after analysis of four or five samples. DNA ploidy heterogeneity, i.e., different samples classified as either DNA euploid or DNA aneuploid in the same tumor was seen in 17%, and DNA index heterogeneity, i.e., tumor populations with different DNA indices (DIs) seen in different samples was 44%. A statistical model defining SPF heterogeneity is proposed. SPF heterogeneity as defined by us was 71%, and as expected the SPF heterogeneity rate increased significantly with increasing number of analysed samples. Four or more samples are needed to detect the most deviant (highest) SPF values. An unrecognized intratumor heterogeneity of DNA ploidy and SPF may partly explain the conflicting results reported in the literature on the above prognostic indicators.

G1-S transition defects occur in most breast cancers and predict outcome.

Cell cycle deregulation is frequently observed in tumors and has moreover been proposed to be a requirement for tumor development. By analyzing the expression of p27 by immunohistochemistry in 100 primary breast tumors and combining the analyses with our earlier characterization of cyclin E, D1, p16, and the retinoblastoma protein (pRB), we have been able to cover the majority of potential G1-S transition defects and observed that 90% of the tumors had alterations in one or several cell cycle regulatory proteins. Considerable variations in protein levels were found among tumors, with low p16 expression as the most common alteration followed by cyclin E or cyclin D1 overexpression, low p27 expression or pRB inactivation in decreasing prevalence. Tumors were grouped according to observed combinations of defects and the proliferative capacity was determined for each group by analyzing Ki-67 labeling index. Low proliferation was observed in tumors with: low p16; high cyclin D1 with normal or high p16 expression; and in tumors without cell cycle defects. Tumors with high cyclin E/low p27 or pRB defects showed higher proliferation. The survival differed noticeably for patients with various combinations of cell cycle defects, and four distinctive clusters were identified showing significantly different breast cancer specific survival (p<0.0001) for both node-positive (p = 0.0006) and node-negative patients (p<0.0001). In summary, we have shown that G1-S transition defects are nearly obligatory in breast tumors and that the specific type of cell cycle defect influences the clinical behavior of the tumor.

Cyclin E dependent kinase activity in human breast cancer in relation to cyclin E, p27 and p21 expression and retinoblastoma protein phosphorylation.

The cell cycle machinery is regulated by cyclin dependent kinases and sets of activating and inhibitory proteins. The G1-S control mechanism is often deregulated in tumours supposedly leading to increased kinase activity, phosphorylation of substrates and subsequent S phase entrance. Increased kinase activity has been proposed to be essential in cell cycle aberrations, but few studies have actually shown enhanced kinase activity related to specific cell cycle defects in primary tumours. In the present study we have determined the cyclin E dependent kinase activity (cyclin E(kinase)) in 59 primary breast cancers, using an H1-kinase assay, and related the activity to the expression of cyclin E, p27 and p21. In a subgroup of 48 tumours, we further characterized the association between cyclin E(kinase), in vivo phosphorylation of the retinoblastoma protein (pRb) and proliferation. The cyclin E(kinase) correlated significantly with cyclin E content and inversely with p27 and p21 expression. P27, but not p21, was associated with low cyclin E(kinase) in specimens with normal/low levels of cyclin E. At elevated cyclin E levels, suppression of cyclin E(kinase) seemed to require high levels of both p21 and p27. The cyclin E(kinase) correlated with the phosphorylation status of pRb as well as with proliferation. Surprisingly, pRb phosphorylation did not correlate with proliferation. Our results support that pRb is a substrate for cyclin E(kinase) in primary breast cancer and that deregulation of cyclin E and p27 act through increased CDK-kinase activity, but cyclin E associated events beside pRb phosphorylation might be rate-limiting for entrance into S phase.

Serum antibodies against p53 in relation to cancer risk and prognosis in breast cancer: a population-based epidemiological study.

To perform an epidemiological evaluation of the predictive value of p53 autoantibodies in breast cancer, we measured antibodies against p53 in serum samples from 165 breast cancer patients in comparison with serum samples from 330 healthy controls, selected from the same population as the cases and matched for age, sex and specimen storage time. Median age of patients was 51 years (range 25-64 years). Presence of serum p53 autoantibodies was analysed by enzyme-linked immunosorbent assay (ELISA) and confirmed by Western blotting. The lower ELISA reactivities were similar for cases and controls, but presence of high-level reactivity was more common among cases than among controls [odds ratio (OR) 9.03, 95% confidence interval (CI) 2.40-50.43]. Presence of Western blot-detected p53 autoantibodies had a very similar association (OR 10.8, CI 3.0-59.4). Among the cases, we also studied whether there was any correlation between level of anti-p53 antibodies and stage of the disease or survival. There was no significant correlation between presence of antibodies and stage of the disease. There was a significant negative correlation between presence of p53 antibodies and survival (P = 0.003). A stepwise multivariate Cox regression analysis showed that T-stage, age and presence of anti-p53 antibodies were significant independent prognostic variables, with a dose-dependent negative effect on survival for all three variables. We conclude that presence of anti-p53 antibodies are of significance both for the risk of having breast cancer and the risk of dying from breast cancer.

Telomerase activity is associated with cell cycle deregulation in human breast cancer.

Deregulation of the cell cycle by abnormal expression of one or several cell cycle regulatory proteins is a common finding in malignant tumors and might be a prerequisite for cancer development. Telomerase activity is an immortalization marker that is found in most cancers and for which an association with an active cell cycle has been implicated. In the tissue of 106 human breast carcinomas, we analyzed the relationship between telomerase activity levels and defects in the cell cycle machinery with a focus on the retinoblastoma protein (pRB) pathway(s). The fraction of telomerase-positive tumors was 85%, and large differences in telomerase activity were found. Overexpression of cyclin D1 and/or cyclin E, in combination with a normal pRB, was a typical feature of tumors with high telomerase activity levels. Down-regulation of p16INK4 was not related per se to telomerase activity, but tumors with low p16INK4 in combination with cyclin D1 or E overexpression demonstrated high activity. Tumor cell proliferation, determined by Ki-67 expression, correlated significantly to telomerase activity levels. There was, however, not a strict association between proliferation rate and telomerase activity, because tumors with inactivated pRB had the highest Ki-67 fractions but intermediate telomerase activity. Also, cyclin D1 overexpression was associated with high telomerase levels without an increase in tumor cell proliferation. The present study indicates that telomerase activation occurs preferentially in breast cancers with certain cell cycle regulatory defects and that telomerase activity levels may depend on the specific defect(s).

Linell classification of breast cancer morphology compared to histologic grading, S-phase fraction and DNA-ploidy.

One hundred and fifty-eight histologically verified mammary carcinomas with known mammographic doubling time (DT) were studied with special emphasis on a morphologic classification proposed by Linell et al. [8, 12, 14, 15]. The hypothesis that Linell classification of ductal carcinomas into comedo, tubuloductal and tubular carcinomas is easy to perform with small inter-observer variations, was not fully confirmed. The Linell classification was found to correlate well with conventional WHO malignancy grading, S-phase fraction and DNA-ploidy. The Linell classification also correlated to surgical stage, lymph node status and DT, but not at all to tumour size. Using distant disease-free survival as an endpoint, the Linell classification gave prognostic information comparable to conventional histologic grading, seeming to be a simple, cheap and reliable method well worth trying on a larger scale.

Deregulation of cyclin E and D1 in breast cancer is associated with inactivation of the retinoblastoma protein.

Inactivation of the retinoblastoma protein (pRB) by mutations or abnormal phosphorylation is a mechanism by which tumour cells can subdue normal growth control. Among molecules involved in control of pRB phosphorylation, cyclin D1 and E have been found to be deregulated and overexpressed in various types of cancers. In order to study the cell cycle regulatory mechanisms in breast cancer, we have analysed the protein expression of cyclin D1 and E in 114 tumour specimens from patients with primary breast cancer using Western blotting. Twenty-five out of 34 tumours with overexpression of cyclin E showed uniform low cyclin D1 expression, and by immunohistochemical analysis of pRB we present evidence for the existence of pRB defects in approximately 40% of these tumours in contrast to no pRB defects in the other group of tumours. This result was supported by a high protein expression of the cyclin-dependent kinase inhibitor p16 in 44% of the tumours with high cyclin E and low D1 expression, and all immunohistochemical pRB defect tumours showed a high p16 protein level. Additionally, an abnormal low pRB phosphorylation in relation to a high proliferative activity and loss of heterozygosity of the retinoblastoma susceptibility gene locus were found in all but one tumour with immunohistochemical defect pRB. Interestingly, tumours with high cyclin E and low D1 expression were generally oestrogen receptor negative suggesting a role for cell cycle regulators in the mechanisms leading to oestrogen independent tumour growth. Furthermore, the prognosis differed markedly for the patients in the various groups of tumours, indicating that the heterogeneous nature of breast cancer pathogenesis and the clinical course in part could be explained by different and distinctive sets of cell cycle defects.

Cyclin E overexpression, a negative prognostic factor in breast cancer with strong correlation to oestrogen receptor status.

Cyclin E is a G1 cyclin which has been proposed to be one of the key regulators of the important G1/S transition, and could consequently be a potential deregulated molecule in tumours. Recently, it has been observed that cyclin E is overexpressed in a variety of malignancies including breast cancer and that several isoforms of the protein exists. In this study we have characterised the cyclin E expression in 114 tumour specimens from patients with primary breast cancer using Western blotting. Various expression of cyclin E was observed among tumours and a group of 27 patients out of 100 patients with stage I-III disease, identified as having tumours with high cyclin E levels, had a significantly increased risk of death and relapse from breast cancer (P = 0.0002 and P = 0.015 respectively). Even in the subgroup of axillary node-negative patients the cyclin E level was of prognostic importance. There was also a strong association between cyclin E expression and oestrogen receptor status (P < 0.00001), and tumours with high cyclin E expression were in general oestrogen receptor negative, suggesting a potential role for cyclin E in mechanisms responsible for oestrogen-independent tumour growth.

Carboxylic ester hydrolase and amylase in ischemic pancreatitis in the guinea pig.

The observation that an elevated level of pancreatic carboxylic ester hydrolase (CEH) in serum is a more sensitive and specific marker of acute pancreatitis than is elevated serum amylase activity prompted us to explore whether these findings could be confirmed in an experimental model and, if so, to find the explanation behind this difference. We therefore developed a model for ischemic pancreatitis in the guinea pig and a sandwich enzyme-linked immunosorbent assay for determination of CEH in this species. There was a strong correlation between duration of ischemia and severity of pancreatic inflammation and between severity of inflammation and serum CEH level. In contrast, serum amylase was elevated only in animals with the most severe grade of inflammation. Amylase was, however, increased in urine in animals with mild inflammation, but the level did not increase with severity of inflammation. Only one of 31 animals had detectable CEH in urine. In animals with intermediate serum CEH levels the serum and biliary concentrations correlated, indicating that CEH may be cleared by the liver. Amylase was detectable in bile only in animals with high serum levels. The results confirm our observations made in previous clinical studies. A likely explanation for differences in serum levels of CEH and amylase is clearance from the circulation at different rates and, at least partly, via different routes, e.g., the liver and kidney, respectively.

Breast carcinoma growth rate described by mammographic doubling time and S-phase fraction. Correlations to clinical and histopathologic factors in a screened population.

BACKGROUND: In a retrospective study, correlations among mammographic doubling times (DT), clinicopathologic prognostic factors, and cytometric predictors were examined. METHODS: One hundred fifty-eight patients with the possibility to calculate mammographic tumor DT were selected and the tumors were histologically reexamined and flow cytometric analysis for ploidy and S-phase fraction (SPF) was performed. RESULTS: The tumors were Stage I in 68%, and 45% were detected by mammographic screening. DT ranged from 0.6 months to an indefinite time (median, 9.0 months). Short DT was significantly correlated to large tumor size (P = 0.01) and advanced pathologic tumor stage (P = 0.016), but there was no correlation between DT and histologic grade. Ploidy analysis indicated that there were 57% aneuploid and 7% tetraploid tumors. There was a significant overrepresentation of euploid tumors among tumors smaller than 10 mm (P = 0.02). Ploidy was correlated to histologic grade (P less than 0.001) and DT (P = 0.009). SPF was calculated in 122 cases. SPF correlated significantly with pathologic stage (P = 0.002), tumor size (P = 0.037), histologic grade (P = 0.001), the presence of axillary lymph node metastases (P = 0.046), DT (P = 0.02), and DNA ploidy (P less than 0.001). Compared with interval carcinoma, screening-detected carcinoma showed favorable characteristics concerning size, stage, DT, ploidy, and SPF but not regarding histologic grade and axillary lymph node metastases. CONCLUSIONS: DT shows great variations. Factors related to tumor biology (i.e., DT, DNA ploidy, and SPF) are strongly correlated with one another, but they have no correlation with axillary lymph node metastases. Cancer detected by screening is discovered at an early stage and shows favorable characteristics concerning DT, ploidy, and SPF.

Mammographic growth rate, DNA ploidy, and S-phase fraction analysis in breast carcinoma. A prognostic evaluation in a screened population.

BACKGROUND: The authors examined prognostic factors in 158 cases of breast carcinoma with known mammographic tumor volume doubling times (DT). METHODS: The tumors were retrospectively reexamined histologically and flow cytometric analysis of DNA ploidy and S-phase fraction (SPF) was performed on archival paraffin-embedded material in each case. Life tables and Cox multivariate analyses were used for statistical evaluation of prognostic factors. RESULTS: In univariate analysis of survival data, clinical and pathologic stage, histologic grade, the presence of axillary lymph node metastases, and SPF were significant prognostic predictors, but mammographic DT and DNA ploidy were not. SPF also contributed prognostic information in the subgroup of carcinoma cases detected by screening. In a Cox multivariate analysis, SPF, the presence of axillary lymph node metastases, and Stage II-III disease (as opposed to Stage I disease) were independent significant predictors of survival. In univariate analyses of distant disease-free survival, clinical and pathologic stage, tumor size, histologic grade, the presence of involved axillary nodes, DT, and SPF all were significant prognostic factors. CONCLUSIONS: SPF, stage, and lymph node status were important prognostic factors in this patient material with predominantly small and node-negative breast carcinomas, whereas DNA ploidy and mammographic DT provided less prognostic information. The prognosis of carcinoma detected during screening did not differ significantly from that of breast carcinoma discovered otherwise in this selected patient group.

Static and flow cytometric DNA analysis compared to histologic prognostic factors in a cohort of stage T2 breast cancer.

DNA analysis with static and flow cytometry was performed on archival smears and tissue sections in 99 patients with T2 breast cancer (Stage II). Tumour size, histologic grade and axillary node metastases were significant prognostic predictors. Static cytometry revealed 63% aneuploid tumours, and ploidy was significantly correlated to histologic grade and survival. DNA measurements obtained by static and flow cytometry were strongly correlated. According to flow cytometry 53% of the tumours were aneuploid. Flow cytometric DNA analysis correlated to histologic grade and survival and gave prognostic information among the lymph-node negative patients. Ploidy seems to be a significant, although not an independent prognostic indicator for T2 breast cancer.

Cystadenocarcinoma of the pancreas 9 years after a pancreatic cyst operation: report of a case.

A patient case with cystadenocarcinoma of the pancreas in a middle-aged woman is described. Nine years prior to the pancreatic malignancy she underwent cystogastrostomy due to a pancreatic cyst presumed to be of non-neoplastic nature. The malignancy occurred at the site of the previous cystogastrostomy and was radically resected. Five years later the patient was well without signs of recurrence. The possibilities of incorrect primary diagnosis or development of a rare pancreatic malignancy in a previous cystogastrostomy are discussed.

Prognostic factors in locally advanced breast cancer (T3, T4) with special reference to tumor cell DNA content.

The prognostic value of DNA analysis was studied retrospectively in 91 patients with locally advanced breast cancer (T3, T4) and a follow-up time of 3-7 years. Tumor cell DNA analysis was performed by static cytometry on aspiration biopsy specimens in 42 cases and on tissue sections in 49 cases. The tumors were classified as euploid or aneuploid. Sixty-four percent of the tumors were aneuploid. DNA pattern correlated significantly to histologic grade and axillary perinodal growth and also to survival. DNA pattern, histologic grade and axillary node metastases correlated significantly to disease-free survival (DFS). In this series of patients with locally advanced breast cancer DNA determinations gave important prognostic information.